Method of enhancing the rate of transfection of cells

ABSTRACT

The invention relates to a method of enhancing the rate of transfection of cells, a method of inserting genetic material into a cell in gene therapy and to a kit containing the components therefor. The method comprises stimulating the cells to be transfected with a growth regulating agent at about the time of transfection. Suitable growth regulating agents include growth promoting agents more particularly still growth factors such as EGF, FGF and NGF.

The present invention relates to a method of enhancing the rate oftransfection of cells, to a kit containing the components therefore andto applications in gene therapy.

The rate of uptake of DNA into cells, and hence the efficiency oftransfection of cells, is dependent on presenting the DNA in a suitablypackaged form for uptake, traditionally by co-precipitation with calciumphosphate, and the ability of the cells to incorporate the packaged DNAinto their cytoplasm.

A paper by Bottger et al, Biochemical et Biophysica Acta 950 (1988)221-228, describes condensation of vector DNA by chromosomal proteinHMG1. The method resulted in enhanced transfection.

It is an aim of the present invention to provide alternative methods ofenhancing transfection which methods could, as well as being of benefitfor transfection of cells in culture, give rise to applications in genetherapy.

The applicants have determined that increased efficiency of transfectioncan be obtained by stimulating the cell membrane with a growthregulating agent, leading to receptor mediated endocytosis, and that theincrease in the rate of transection is at least as great as thatresulting from enhanced packaging of DNA with HMG1.

According to a first aspect of the present invention there is provided amethod of enhancing the rate of transfection of cells comprisingstimulating the cells to be transfected with a growth regulating agentprior to transfection.

Preferably the growth regulating agent is a growth promoting agent, moreparticularly still a growth factor.

An advantage of using a growth regulating agent is that they can beselected to be specific or non-specific.

Thus, Epidermal Growth Factor (EFG), in spite of its name, willstimulate almost any cells of endothelial or epithelial origin,Fibroblast Growth Factor (FGF) stimulates cells of mesothelial originand Nerve Growth Factor (NGF) has a very narrow specificity for neuronalcells. The specificity is dictated by the presence of specific receptorsfor the growth factors on the cell membrane.

This specificity may be particularly advantageous when it comes toconsider gene therapy applications.

This is because a major problem to be overcome with gene therapy is howto achieve high rates of transfection with some specificity. Somepresent approaches can achieve physical localisation of the deliveredgene, for instance to a specific organ. The methodology of the inventionwould, however, allow the selective targetting of a particular cell typewithin that organ, in addition to enhancing the overall level oftransfection.

Other advantages over prior art methods are the cost benefit and thepossibility of avoiding introducing "foreign" matter (e.g. viral matter)

As well as growth factors, other growth regulating agents such as, forexample, Peanut Agglutinin and Mushroom Lectin could be used.

Lectin Peanut Agglutin is, for example, thought to promote the growth ofcells by interaction with surface glycoproteins. In contrast Mushroom(Agaricus bisporis) Lectin, which shares the specificity of peanutagglutinin, is inhibiting to the growth of cells. Mushroom Lectin isfound to be carried within the cell to the surface of the nuclearmembrane and may possibly carry the DNA with it though the cellmembrane. It is thus envisaged that, for example, a combination of agrowth factor such as EGF with mushroom lectin would allow the enhancingeffects of the growth promotion to be used for gene therapy without thepossible disadvantage of the growth promoting effect in vivo.

In accordance with a further aspect of the present invention there isprovided a method of inserting genetic material into a cell in genetherapy comprising stimulating the cell into which genetic material isto be inserted with a growth regulating agent in the presence of agrowth inhibitor.

According to yet a further aspect of the present invention there isprovided a kit for the methods of the invention comprising at least onegrowth regulating agent; and a buffer.

The Growth regulating agent should be present in an effective amount.For EGF an effective amount has been determined to be 1 μg /ml, althoughother amounts, for example, 0.1 μg to 10 μg may be used.

Optionally the kit also comprises one or more of the following: aprecipitant, for example, CaCl₂ and/or HMG1, a plasmid control, e.g.PSV2neo, a marker e.g. genticin and a set of instructions.

Preferably the growth regulating agent is one or more of EGF, FGF, NGF,PDGF, IGF1, lectin peanut agglutinin and mushroom lectin.

Preferably the buffer is a HBS buffer comprising, for example, 280 mMNaCl, 50 mM HEPES and 1.5 mM Na₂ HPO₄.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the transfection rate for:

1. EGF and HMG1

2. EGF alone

3. HMG1 alone

4. Calcium phosphate (Control), and

5. No Calcium phosphate.

FIG. 2 is a graph showing transfection rates at different concentrationsof FGF. FIG. 2 shows that transfection by DNA is increased by bothacidic and basic FGF.

The invention will be further described by way of example only withreference to the following example.

METHOD

Cells and Growth Regulating Agents.

A rat mammary (Rama) cell line was cultured in Routine Medium(Dulbecco's Modified Eagle's Medium (Gibco, Paisley, UK) supplementedwith 5% foetal calf serum (Biological Industries, Glasgow, UK), 50 ng/mlinsulin, and 50 ng/ml hydrocortisone (Sigma, Pool, UK). The Rama 27 cellline was originally derived form the fast sticking fraction of cellsisolated from a normal rat mammary gland and was defined as fibroblaston the basis of its ability to differentiate to the adipocyte phenotype(Rudland, P. S. et al (1984) J. Cell. Physiol. 120, 364-376). Epidermalgroup factor (EGF) was purified from mouse submandibular glands (Smith,J. A. et al (1984) J. Chromatogr. 305, 295-308).

Transfection

Rama 27 cells were passaged into 50mm dia, plates at a density ofapproximately 0.9×10⁶ cells/plate, and incubated overnight at 37° C. Themedium was changed 3 to 4 hours before transfection. Cells to betransfected were treated with 1 ml of a solution containing 500 μl 2×HBSand 125 μl 1MCaCl₂ and 375 μl H₂ O, and a final concentration of 20μg/ml pSV2neo (Graham, F. L. et al Virology 52, 456, 1973), togetherwith other components as indicated. The plasmid and CaCl₂ were mixedusing a peristaltic pump attached to a sterile siliconised Pasteurpipette. The suspension was allowed to stand for 30-45 min, inverted tomix and added directed to the culture medium. Growth factor, whereappropriate was then added, and the plates were incubated at 37° C. for4 hours. The medium was removed, replaced with normal medium containing100% DMSO, and incubated for 90 sec. DMSO containing medium was removed,the cells were washed with routing medium, and incubated with freshmedium for 24 hours. Cells were plated at a density of 3×10⁵ cells per90 mm plate in medium containing 0.5 mg/ml geneticin initially, andsubsequently, 1 mg/ml (selective medium). Selective medium was changedevery 3 to 4 days during a 3 week period until colonies were clearlyvisible. The number of colonies was then counted. When used, HMG1 had afinal concentration of 200 μg/ml (Bottger et al, (1988) Biochim.Biophys. Acta 950, 221-228) and EGF 10 ng/ml.

Results

                  TABLE 1                                                         ______________________________________                                        Transfecting                                                                            Growth                     Transfection                             Agent     Factor   CaPO.sub.4                                                                             No. of colonies                                                                        Rate (/10.sup.5)                         ______________________________________                                        --        --       +        0        0                                        PSV2neo   --       +        5        1.7                                      PSV2neo   EGF      +        18       6                                        PSV2neo/HMG1                                                                            --       +        16       5.3                                      PSV2neo/HMG1                                                                            --       -        4        1.3                                      ______________________________________                                    

The cells were resistant to geneticin only after treatment with thetransfecting agent, showing that the resistance was caused by thetransfected neo gene.

From Table 1 above it is apparent that the best transfection rate wasachieved when a growth regulator EGF was included.

The results suggest that the growth regulating agents have a significantrole in enhancing the uptake of genetic material (genes plasmids,antisense DNA, oligonucleotides, etc.) into cells in culture or in vivo,presumably by enhancing the rate of infolding of the membrane of thecells.

The method is expected to have the advantage that it will be additive toother methods; e.g. the use of HMG1, which acts by packaging the DNAmore attractively to the cells.

The method should convey a degree of specificity to the mode ofdelivery, in that for each type of tissue or cell, there will be apreference for the response to growth factor. Thus, for example, thegenetic material might be released in a specific tissue by physicalmeans, but directed to a particular cell type within that tissue by theuse of a growth factor specific to that cell type in order to increaseoverall uptake and/or to reduce side effects caused by delivery to thewrong cells.

Because of its non-specific mechanism, the method should also haveapplicability to situations in which it is required to increase theuptake of peptides or other hydrophilic compounds into cells.

In a further study the effect of EGF and HMG 1 on transfection rates wasinvestigated.

The results are shown in FIG. 1 which is a graph showing thetransfection rate for:

1. EGF and HMG 1

2. EGF alone

3. HMG alone

4. Calcium phosphate (Control), and

5. No Calcium phosphate

The results show EGF is significantly better than calcium phosphate (thecurrent product) and that the case of EGF and HMG 1 together produceeven better results.

These results support the applicants belief that EGF and HMG 1 act indifferent ways. The results are compatible with the idea that the growthfactor enhances the rate of uptake in the cells whereas the HMG 1protein binds DNA into a preferable conformation and transports it tothe nucleus.

In yet a further study the effect of both acidic and basic FGF ontransfection rates was investigated. The results are illustrated in FIG.2 which is a graph showing transfection rates at differentconcentrations. FIG. 2 shows that transfection by DNA is increased byboth acidic and basic FGF. Surprisingly the acidic FGF is better thanthe basic FGF because basic FGF is better than the acidic FGF when itcomes to promoting growth of Rama 27 cells.

What is claimed is:
 1. A method enhancing the rate of transfection ofcells comprising stimulating the cells to be transfected with a free andunbound growth factor together with HMG 1 at about the time oftransfection.
 2. A kit for enhancing the rate of transfection of cellscomprising a free and unbound growth factor together with HMG 1, and abuffer.
 3. The kit of claim 2 further comprising a precipitant, aplasmid control, a selective agent, and a set of instructions.
 4. Themethod of claim 1 wherein the growth factor is specific to the cellsbeing transfected.
 5. The method of claim 1 wherein the growth factor isnon-specific to the cells being transfected.
 6. The method of claim 1 inwhich the growth factor is epidermal growth factor (EGF).
 7. The methodof claim 1 in which the growth factor is fibroblast growth factor (FGF).8. The method of claim 7, wherein the FGF is acidic FGF.
 9. The methodof claim 1 in which the growth factor is nerve growth factor (NGF). 10.The method of claim 1 in which the growth factor is platelet derivedgrowth factor (PDGF).
 11. The method of claim 1 in which the growthfactor is insulin like growth factor 1 (IGF 1).
 12. The method of claim1 wherein a growth regulating agent is used in combination with thegrowth factor.
 13. The method of claim 12 wherein the growth regulatingagent is a lectin.
 14. The method of claim 13 wherein the lectin islectin peanut agglutinin.
 15. The method of claim 13 wherein the lectinis a mushroom lectin.
 16. A method of inserting genetic into a cellcomprising stimulating the cell growth into which genetic material is tobe inserted with a free and unbound growth factor together with HMG 1 inthe presence of a growth inhibitor.
 17. The method of claim 1 whereinthe cells are in culture.